Indigenous cellulolytic aerobic and facultative anaerobic bacterial community enhanced the composting of rice straw and chicken manure with biochar addition

Microbial degradation of organic matters is crucial during the composting process. In this study, the enhancement of the composting of rice straw and chicken manure with biochar was evaluated by investigating the indigenous cellulolytic bacterial community structure during the composting process. Compared with control treatment, composting with biochar recorded higher temperature (74 °C), longer thermophilic phase (> 50 °C for 18 days) and reduced carbon (19%) with considerable micro- and macronutrients content. The bacterial community succession showed that composting with biochar was dominated by the cellulolytic Thermobifida and Nocardiopsis genera, which play an important role in lignocellulose degradation. Twenty-three cellulolytic bacterial strains were successfully isolated at different phases of the composting with biochar. The 16S rRNA gene sequencing similarity showed that they were related to Bacillus licheniformis, Bacillus subtilis, Bacillus aerius, and Bacillus haynesii, which were known as cellulolytic bacteria and generally involved in lignocellulose degradation. Of these isolated bacteria, Bacillus licheniformis, a facultative anaerobe, was the major bacterial strain isolated and demonstrated higher cellulase activities. The increase in temperature and reduction of carbon during the composting with biochar in this study can thus be attributed to the existence of these cellulolytic bacteria identified.

Solid waste management is crucial in reducing and eliminating the adverse effect of waste materials on human health and the environment. According to the world bank statistic, the global solid waste generated was 2.01 billion tons in 2016 and is expected to increase by 3.4 billion annually by 2050 1,2 . Effective treatment of these wastes will reduce their hazardous impacts and promotes sustainable development goals. Several technologies such as anaerobic digestion and composting have been developed for treating various solid waste generated from industrial, agriculture and livestock activities. Until now, composting has remained the most acceptable method used to treat animal manure because of its ability to remove pathogens, diminish odor generation, reduce the volume and moisture and improve storage and transportation options.
Composting is a simple biological process in which the organic materials are degraded naturally into stable humus-like substances facilitated by the indigenous microorganisms under aerobic conditions. The process is greatly assisted by microorganisms such as fungi, bacteria and actinomycetes which act as composting agents

Materials and methods
Composting procedure. Chicken manure and rice straw were used as the main components for the composting process. The composting operation and the sampling process were carried out according to the method described by Zainudin et al. 13 . Briefly, the composting process was performed by mixing 150 kg (w/w) of chicken manure and 50 kg (w/w) of rice straw, equivalent to a 2:1 ratio, in a round high-density polyethylene bin with a diameter of 162 cm and height of 132 cm. The height of composting pile was around 100 cm. Composting was performed beneath the shed to prevent the effects of weather variations such as hot and rainy conditions. The rice straw used in this study was collected from the paddy field in Sekinchan, a district located on the west coast of peninsular Malaysia. The rice growers have given their consent for the acquisition of rice straw. The chicken manure was collected from the poultry farm at the Animal Research Center of the Institute of Tropical Agriculture and Food Security, UPM. Twenty percent (w/w) of oil palm empty fruit bunch (EFB) biochar was then mixed with the chicken manure and rice straw. The biochar used in this study was previously produced by Idris et al. 14,15 at a temperature of 600 °C in a pool-type biochar reactor. The biochar characteristics are shown in Table 1. Another set of treatments without the addition of biochar was made as a control. The composting was done twice for each of the treatments due to the limited number of composting bins. The water content was set at 60% with no further adjustment of moisture until the composting process finish. The turning process was carried out every seven days to facilitate the aeration and homogenization of the composting material. The temperature was recorded by inserting the temperature meter probe (DMS-30LCD-1-5, DATEL, USA) at the core of the composting pile. The sample was collected during each of the turning processes. One representative composite sample was taken from a randomized location mainly from the middle and bottom part of the pile. The pH and moisture content of the compost was determined according to the method described by Wei et al. 16 and Zainudin et al. 13 , respectively. Briefly, the pH was measured by mixing one gram of sample with 10 ml of distilled and the aqueous solution was then measured using a pH meter. The moisture content was measured by drying 10 g of the sample at 105 °C in a moisture analyzer (MX/MF-50 Moisture Analyzer, USA). The macronutrients: carbon (C) and nitrogen (N) were determined using LECO TruMac CNS elemental analyzer (MI, USA) while micronutrients: Phosphorus (P), Potassium (K), Calcium (C), Magnesium (Mg), Iron (Fe), Zinc (Zn), Copper (Cu) and Manganese (Mn) were detected using Inductively Coupled Plasma (ICP-OES, Perkin Elmer, USA). The analysis was performed based on a dry basis and each sample was measured in three replicates. All experimental methods were performed in accordance with relevant guidelines and regulations.
High-throughput 16S rRNA gene analysis. DNA

Isolation of mesophilic and thermophilic cellulolytic bacteria. The isolation of cellulolytic bacteria
was done according to the method described by Zainudin et al. 17 . One gram of sample was added into the 50 ml tube containing 10 ml of Luria Bertani (LB) broth. The samples were serially diluted and were cultured onto LB and Tryptic Soy (TS) medium agar plate. The plates were then incubated at mesophilic (37 °C) and thermophilic (50 °C) temperatures for 12 to 24 h. The colonies grown on the agar plate were selected and streaked onto TS and LB medium agar plates containing 0.2% carboxymethyl-cellulose (CMC). The bacteria harboring the cellulolytic activity were identified as the strains that produce a clear zone around the colony. The isolated bacteria colony was repetitively sub-cultured to obtain a single pure culture.
Cellulolytic activity assay. The enzymes activities of isolates were evaluated by point-inoculating the culture onto the surface of the TS and LB agar plate containing 0.2% CMC and incubated overnight at 37 °C and 50 °C, respectively. The cellulolytic activity was expressed as the substrate hydrolysis index. The index was calculated as the ratio between the diameter of the hydrolysis zone around the colony and the diameter of the colony in millimeter using the formula as follow: Identification of isolated strains. The identification of the isolates was done based on 16S rRNA gene sequencing. The Polymerase Chain Reaction (PCR) was performed based on the PCR colony method as described by Zainudin et al. 17 . A pair of universal primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT -3') targeting the 16S rRNA gene was used as forward and reverse primers. The amplification of the 16S rRNA gene was conducted in 50 μl of PCR Master Mix kit (QIAGEN) mixture containing 25 μl dNTP's, 1.5mM MgCl 2 buffer, DNA polymerase, 0.5 μl of each forward and reverse primers. The RNase-free water was added until the final volume reaches 50 μl. Thermocycling was set up according to the conditions provided by the kit protocol as follows: 3 min of initial denaturation at 94 °C, 0.5 min of denaturation at 94 °C, 0.5 min of primer annealing at 60 °C, 1 min of primer extension at 72 °C and final elongation at 72 °C for 10 min. The PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized using Gel red staining on a UV transilluminator. The PCR products were purified using Nucleospin PCR clean-up (Machery-Nagel, GmbH & Co., Germany) according to the manufacturer's instructions before sequencing. The PCR products were then sequenced and the 16S rRNA sequences data were compared with those of other know species in the genebank database (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi).
Cellulolytic index = Diameter of clear zone (mm) − Diameter of bacteria colony (mm) Diameter of bacteria colony (mm)

Results and discussion
Composting characteristics. Table 2 shows the main characteristics of the composting process with and without the addition of biochar. The final composting process with biochar showed a significant (P<0.05) reduction of carbon, representing 19% of the initial C content, whereas in the composting without biochar, carbon reduction was only 8%. Both piles showed the escalation of the N content at the final stage of the composting process. However, the addition of biochar did not show any significant reduction (P>0.05) in the amount of N, supporting the hypothesis that biochar retains nitrogen in the composting mixture through the absorption of the nitrogenous compounds such as ammonia and ammonium onto the surface of biochar. Previous research has shown that composting with biochar reduces NH 3 emissions while increasing NO 3 concentrations when compared to composting without biochar. According to Zainudin et al. 13 , the biochar addition to the composting pile improves the nitrification process, in which NH 4 + is transformed into NO 3 by nitrifying bacteria. Furthermore, the oxidation of aromatic and carbonyl groups led to the creation of a positive and negative charge on the surface of biochar, promoting NH 4 + and NO 3 adsorption 18 . The overall decrease in C content was higher in the biochar treatment pile than in the composting without biochar. In this study, composting was conducted by combining the chicken manure and rice straw at the ratio of 20:1 ratio, or C/N:15.6. According to Zhu 19 and Zhou 20 , aerobic composting of swine manure with rice straw, edible fungal residue and rice bran at a low C/N ratio improves the maturation rate and increases organic matter degradation as compared to high C/N ratio composting. This finding is also in accordance with a previous study, which found that the dissolved organic carbon (DOC) was lower in biochar amended compost than without biochar due to the increased microbial activity 18 . It has been suggested in previous studies that adding biochar reduces the bulk density, which subsequently improves the aeration of the composting pile. The improved aeration promotes microbial proliferation and activity,thus, enhancing nutrient mineralization throughout the composting process. Thus, our finding supports the evidence of previous studies which reported that biochar improves organic matter degradation, hence, reducing the carbon content. Aside from the higher C reduction, the macro-and micronutrients such as phosphorus, kalium, calcium, magnesium, and zinc in composting with biochar were also higher than that of control composting. This could be due to the higher inorganic nutrient contents in the EFB biochar 14,15 . In addition, the organic coating forms on the outer and inner pore surfaces of biochar particles promotes the nutrient retention of the co-composted biochar 21 . The temperature of the composting process lasted for 14 days in control composting and 18 days in composting with biochar (Fig. 1a). The extended thermophilic phase is common for this organic material because rice straw contains recalcitrant compounds that are difficult for microorganisms to degrade, thus lengthening the composting process. However, the maximum temperature of the pile was higher in composting with biochar (74 °C) than in composting without biochar (62 °C), indicating that the addition of biochar enhanced the microbial activities, especially for the lignocellulose degradation. A study conducted by Huang et al. 22 indicated that composting of pig manure and sawdust with an initial C/N of 15 resulted in a gradual rise in temperature, lower maximum temperature, and shorter thermophilic phase. However, it is interesting to demonstrate in our study that higher maximum temperature, rapid temperature increase, and longer thermophilic period can be achieved in composting with biochar at a low C/N ratio, implying the function of biochar in promoting the composting process as a result of higher organic matter degradation by microbes. Table 2. Characteristics of initial mix and final product of rice straw and chicken manure composting with and without biochar addition. The symbol (*) indicates a significant difference at P ≤ 0.05 between the samples. www.nature.com/scientificreports/ The pH of the pile increased, whereas the moisture decreased as the composting progressed (Fig.1b,c). The results corresponded with a previous study which reported that the biochar addition increased the pH of the composting pile, which was due to the alkalinity properties of the biochar 23 . The physicochemical properties suggest that the enhancement of composting process could be due to the addition of biochar which improves the microbial activities for organic matter degradation. Therefore, in this study, we attempt to identify the bacterial community structure using high-throughput 16S rRNA gene sequencing and isolation of cellulolytic bacteria to evaluate further their relevance in improving the composting process with biochar.
Bacterial community structure. The high-throughput 16S rRNA gene sequencing was performed to clarify detailed information about the bacterial population during both of the composting processes. The heatmap analysis showed that the composting with biochar was generally dominated by the Thermobifida, Nocardiopsis, Compostibacillus, Ammonibacillus, Sinibacillus, Bacillus, Truepera, Halomonas, Pseudofulvimonas, respectively (Fig. 2). These bacteria were often discovered during agricultural waste composting 13,24-26 . During the thermo- www.nature.com/scientificreports/ philic phase, the most prevalent taxa were Sinibacillus, Bacillus, Compostibacillus and Thermobifida. However, the abundance of Sinibacillus, Bacillus, and Thermobifida decreased as the composting progressed. In contrast, the abundance of Nocardiopsis increases when the composting enters the mesophilic and mature phases. Thermobifida and Nocardiopsis were highly abundant in the composting with biochar as compared to composting without biochar. In addition, the abundance of Bacillus, Sinibacillus and Compostibacillus during the thermophilic phase of the composting with biochar was higher than that of composting without biochar. These bacteria were able to live at the high temperature of the composting pile due to their ability to generate endospores. A previous study showed that members of Bacillus and Sinibacillus were mainly responsible for the degradation of highmolecular-weight of organic matter throughout the composting process 24 . Moreover, Compostibacillus which was previously isolated from sludge composting has been found as a facultative anaerobe and is capable of growing at temperatures as high as 60 °C 27 . Facultative anaerobes can grow and metabolize in aerobic and anaerobic environments although they favor oxygen-rich conditions 28 . Therefore, the increased abundance of Compostibacillus supports the finding of previous studies that the addition of biochar enhanced the aeration of the composting pile, thus, encouraging the development of bacteria including facultative anaerobe. Truepera, Halomonas and Pseudofulvimonas genera which are known as nitrifying and denitrifying bacteria 13 showed an increasing trend as the composting entered the mesophilic and maturing stages. These bacteria were found to be highly dominant in the composting with biochar.
Cellulolytic bacteria. Composting with biochar resulted in a greater reduction of carbon content, which might have been assisted by the presence of cellulolytic bacteria. As a result, in this study, we aim to isolate as many cellulolytic bacteria as possible from the composting with biochar. The isolation of cellulolytic bacteria was also done from the composting without biochar for comparison. The results showed that the number of isolated cellulolytic bacteria was greater in composting with biochar than in composting without biochar (Table 3). Twenty-eight of the isolated strains were found to be more than 99% identical to known cellulolytic bacteria. These strains were closely related to Bacillus licheniformis, Bacillus subtilis, Bacillus aerius, Bacillus haynesii, all of which were known to exhibit cellulolytic activity and have been involved in the lignocellulose degradation process [29][30][31] . Of these cellulolytic strains, B. licheniformis was the primary species isolated from the composting with biochar. The results corresponded with 16S rRNA sequencing data, which indicated that Bacillus was among the dominant bacteria detected throughout the composting process. B. subtilis had previously been isolated from soil and compost and was known to demonstrate cellulolytic activities 32,33 .  www.nature.com/scientificreports/ This bacterium has been widely used in various kinds of applications including cellulase production for saccharification and as an inoculum to enhance the composting process 34,35 . B. aerius is a thermophilic cellulolytic bacterium that was previously isolated from lignocellulosic waste and hot-spring sediment 30,36 . This bacterium plays a major role in the hydrolysis of lignocellulose and has been found to produce highly thermostable cellulases 37 .

Relationships between physicochemical properties and bacterial community. Since compost-
ing is a microbially-mediated process, understanding the relationship between microbe and physicochemical properties of the compost will substantially improve the efficiency of the composting process and the quality of its end-product. Therefore, a principal component analysis (PCA) was performed to explain the relationship between the dominant bacteria community, particularly cellulolytic bacteria, and physicochemical characteristics during the composting process with biochar. The results showed that Thermobifida and Nocardiopsis exhibited positive correlations with N content and pH, respectively (Fig. 3). In contrast, they showed negative correlations with C content, indicating their important roles in organic matters degradation, particularly the lignocellulosic materials, thus, reducing the C content as the composting progressed. Thermobifida and Nocardiopsis genera were known as cellulolytic bacteria and had been involved in the lignocellulose degradation 17,25 . They were important cellulolytic actinobacteria capable of generating a variety of hydrolytic enzymes for lignocellulose degradation, including exoglucanase, endoglucanase, β-glucosidase and xylanase. The majority of the www.nature.com/scientificreports/ enzymes produced by these bacteria were very stable at a broad range of pH and temperature 38,39 . These bacteria are aerobic, gram-positive and spore-forming bacteria and these traits are critical for their survival in harsh environments. Similar to Thermobifida and Nocardiopsis, the cellulolytic B.licheniformis, also showed an inverse correlation with the C content, indicating its function in the degradation of the lignocellulose matrix. On the other hand, B.licheniformis positively correlated with pH but negatively correlated with moisture content and temperature. B. licheniformis is a facultatively anaerobic, gram-positive bacterium that has been isolated in a wide range of temperature environments 40 . In this study, B. licheniformis was successfully isolated at mesophilic and thermophilic temperatures, indicating that this bacterium can grow and thrive in both conditions 41 . According to He et al. 42 , B. licheniformis would grow fast and require less doubling time in the presence of oxygen. As suggested earlier, the purpose of adding biochar into the composting mixture was to lower the bulk density of the components, hence, enhancing the oxygen penetration to the interior part of the pile. The high-water content of the composting without biochar (Fig. 1c) might saturate the system and reduce the oxygen penetration, thus, decreasing the B. licheniformis population. As a result, the reduced moisture content of the composting with biochar enhanced the compost environment for the B. licheniformis development. Previous studies reported that the micro-aerobic pretreatment of lignocellulose corn straw and microalgal biomass increased the activity of the hydrolytic enzymes such as cellulase and encouraged the development of facultative anaerobic bacteria 42,43 . Furthermore, B. licheniformis isolated from bovine rumen had the highest cellulose-degrading activity under micro-aerophilic conditions 44 . This corresponds with our earlier discussion that the increasing number of cellulolytic facultative anaerobe B. licheniformis could be attributed primarily to adequate oxygen supplementation for this bacterium to thrive as a result of the large surface area and porosity of biochar, which produces the micro-aerobic conditions inside of the composting pile. In addition, Truepera, Halomonas, Pseudofulvimonas also showed a negative correlation with C content, indicating that the increased abundance of these genera could be attributed to the consumption of readily utilizable organic fraction generated as a result of the organic matter degradation during the thermophilic stages, hence, reducing the carbon content towards the end of the composting process. Overall, the findings of this study suggest that the enhancement of the composting with biochar was due to the abundance of cellulolytic bacteria detected, which may have led to higher organic matter degradation especially the lignocellulosic material throughout the composting process. www.nature.com/scientificreports/

Conclusion
The treatment with biochar showed an increase in the temperature of composting pile and reduced the carbon content while retaining a considerable amount of micro-and macronutrients as compared to composting without biochar. High-throughput 16S rRNA gene analysis demonstrated that the thermophilic stage was dominated by cellulolytic bacteria related to the Thermobifida genus, while the Nocardiopsis genus dominated the mesophilic and maturing stages. Isolation of cellulolytic bacteria also indicated that the strains related to the facultative anaerobe B. licheniformis, B. subtilis, B. aerius and B. haynesii strains were identified at different stages of the composting process. The finding of this study showed that the cellulolytic bacterial community was significantly correlated with changes in physicochemical properties, notably the C content, suggesting that their existence might be the reason for the composting process's improvement. www.nature.com/scientificreports/